Dental caries is one of the most prevalent chronic infections in humans. It is caused by acid production from fermentation conducted by acidogenic bacteria that colonize the tooth surface. Intake of fermentable carbon by the host promotes acid generation, resulting in tooth demineralization, which is symptomatic of dental caries. Establishment of the acid-producing biofilm that leads to caries is initiated by Streptococcus mutans, which produces adhesive proteins and glucans required for biofilm development. Although it contributes to pH reduction, S. mutans is sensitive to extremes of pH. Such extremes are rarely encountered in the oral environment, as normal saliva has a pH range from 6.0 to 7.8, with stimulated saliva flow having a pH from 7.4- 7.8. While much research has been devoted to understanding how S. mutans withstands low pH, there are few studies that have targeted the response of S. mutans to alkaline conditions. Quite unexpectedly, a deletion of the adhC gene, encoding the lipoylated E2 subunit of acetoin dehydrogenase (Adh), confers acute sensitivity to pH of ~7.5, a pH value commonly observed in human saliva. Mutations that render defective production of the other adh operon products (E1, E3, and LplA, the lipoyl ligase) also confer sensitivity to modest elevations in pH (pH 7.2-7.6). The adh/lplA mutants also exhibit defects in carbohydrate uptake and/or consumption and the adhD null mutation confers a severe defect in biofilm formation when sucrose is present. The finding raises the possibility of targeting specific functions in S. mutans in order to sensitize the bacterium to the ambient pH of the human oral cavity while also compromising metabolic operations within the oral pathogen. The exploratory, hypothesis-generating project proposed herein will identify the factors associated with Adh- dependent alkaline tolerance. Suppressor mutations that overcome the alkaline sensitivity of the adhC mutation have been isolated and will continue to be uncovered. Such mutations will identify genes that potentially operate within the network that Adh functions to render cells resistant to elevated pH. The lipoyl cofactor attachment sites of encoded in adhC and adhD will be mutationally inactivated and the effect on alkaline sensitivity will be tested to assess the importance of Adh redox chemistry in alkaline tolerance. Micromolar Zn2+ concentrations, which inhibit 2-oxo acid dehydrogenases, will be used to examine test the Adh catalytic requirement for pH tolerance. A genomic Tn-seq experiment will be undertaken to identify genetic loci that function in alkaline tolerance. These mutations will be combined with the adhC suppressor mutations to determine if the identified genes? functions are related to that of Adh-dependent alkali resistance. The adh mutants and those identified in the mutant screens will be tested for fitness in mixed cultures with commensal, arginolytic competing species, S. gordonii or S. sanguinis, which are known to cause pH elevation in plaque biofilms. These experiments will be performed with planktonic cultures and mixed-species biofilms. The project will generate potential targets for interfering with S. mutans proliferation in the human oral environment.